RNA Interference (RNAi) promises to become a powerful approach to rational drug design. One significant drawback to RNAi is that its effect on gene expression is rarely 100% potent. This limitation, observed widely, is problematic when a complete cessation of gene expression is desired or required. A major goal of this proposal is to develop targeted strategies to enhance the efficacy of gene silencing during commercial RNAi applications. Our goal is to develop small molecule mimetics and RNA-based compounds that target core pathways important in determining RNAi efficacy. Application of such compounds in conjunction with an RNA-based drug or treatment will boost performance of that drug or treatment. Our initial target was discovered as a Drosophila mutant that exhibits enhanced RNAi activity - three times stronger than normal - without any detectable side-effects. This occurs because the mutant lacks an important inhibitory gene that naturally limits RNAi efficacy. The inhibitor specifically attenuates one step in the assembly of short interfering RNAs (siRNAs) with the complex that degrades target mRNAs. The existence of such a gene argues that natural physiological mechanisms restrict RNAi efficacy. Moreover, highly conserved homologous genes exist in all sequenced vertebrate genomes, including humans. It is our hypothesis that these genes also restrict RNAi activity in these species. Thus, blocking the inhibitor's activity should improve RNAi efficacy. We propose to test the feasibility of developing siRNAs that block the inhibitor and thereby boost performance of an administered RNAi drug or treatment. We will evaluate the RNAi activity in mouse cell lines mutant for the inhibitor. We will evaluate the RNAi activity of human and mouse cell lines treated with siRNA or shRNA to knockdown the inhibitor. We will evaluate the effect of inhibitor depletion (by siRNA) on boosting RNA-based therapies such as killing prostate tumor cells and blocking influenza virus replication. Development of compounds that boost RNAi performance in general will have significant impact on public health. [unreadable] [unreadable] [unreadable] [unreadable]